Tumor is a result of abnormal, incontrollable and disordered cell proliferation including excessive abnormal cell proliferation. When a tumor exhibits destructive proliferation, infiltration and metastasis, it is classified as a malignant tumor. In particular from the view point of molecular biology, a tumor is considered as a genetic disease caused by mutation of a gene. To treat malignant tumors, three treatment methods which are surgical operation, radiotherapy and chemotherapy have been conducted either separately or together. Particularly, surgical operation is a method to eliminate most of pathogenic tissues, which is thus very effective to remove tumors growing in the breast, colon and skin but not so effective to treat tumors in spine and dispersive tumors.
Radiotherapy has been performed to treat acute inflammatory, benign or malignant tumors, endocrine disorders and allergies, and it has been effective to treat such malignant tumors resulted from abnormal rapid cell division. However, the ratio therapy carries serious side effects such as functional disorder or defect of normal cells, outbreak of cutaneous disorders on the treated area and particularly retardation and anostosis in children.
Chemotherapy is a method to disturb duplication or metabolism of cancer cells, which has been performed to treat breast cancer, lung cancer and testicular tumor. The biggest problem of this treatment method is the side effect carried by systemic chemotherapy. Side effects of such chemotherapy are lethal and thus increase anxiety and fear for the treatment. One of the representative side effects of chemotherapy is dose limiting toxicity (DLT). Mucositis is one of examples of DLT for various anticancer agents (antimetabolic agents such as 5-fluorouracil and methotrexate, and antitumor antibiotics such as doxorubicin). Most cases of side effects require hospitalization or at least need pain killers. So, side effects by chemotherapy and radiotherapy are such as are serious matters for the treatment of cancer patients.
In the meantime, gene therapy is based on the DNA recombination technique, which is the method to insert a therapeutic gene into cancer patient cells to correct gene defect or to endow a novel functions to disordered cells to treat or prevent various genetic diseases caused by mutations diseases, infective of genes, diseases, cancer, autoimmune cardiovascular diseases, etc. More particularly, gene therapy is a method to treat the said diseases by inducing intracellular expressions of normal proteins or therapeutic target proteins by conveying a therapeutic gene into a target organ. Gene therapy has an excellent selectivity, compared with other treatment methods using drugs and can be applied for a long term with improved treatment effect on difficult diseases. To enhance the therapeutic effect of gene therapy, gene transfer technique is essential for the realization of high efficient gene expression in target cells.
A gene carrier is a mediator for the insertion of a therapeutic gene into a target cell. A preferable gene carrier is the one that is not harmful for human, can be mass-produced and has ability to transmit a therapeutic gene effectively and induce constant expression of the therapeutic gene. Thus, gene transfer technique is a key factor for gene therapy and representative gene carriers most wanted for gene therapy so far are exemplified by viral carriers such as adenovirus, adeno-associated virus (AAV), and retrovirus; and non-viral carriers such as liposome and polyethyleneimine.
It is one of the strategies of gene therapy to control tumor cells by using a tumor suppressor gene, a tumor-specific killer virus, a suicide gene and an immunoregulation gene. Particularly, the method using a tumor suppressor gene is to treat cancer by conveying the original form of a tumor suppressor gene such as p53, which is deficient or mutated in many cancer patients. The method using a tumor-specific killer virus is to treat cancer by conveying a virus gene carrier that can be proliferated selectively in tumor cells into cancer patients by taking advantage of the activity of a tumor suppressor gene transformed in cancer tissues. The basic strategy of the above two methods is to kill tumor cells directly. In the meantime, the method using a suicide gene is to induce suicide of tumor cells by inserting sensitive genes such as HSK-TK. The method using an immunoregulation gene is to treat disease indirectly by stimulating T-cell mediated tumor cell recognition by delivering a gene increasing antitumor immune response such as interleukin 12 (IL12), interleukin 4 (IL4), interleukin 7 (IL7), γ-interferon and tumor necrosis factor (TNF) or by inducing apoptosis by interrupting tumor inducing proteins.
In relation to gene therapy among various attempts to treat cancer, the present inventors selected ANT (adenine nucleotide translocator) as a target gene to develop an effective safe anticancer agent.
ANT (adenine nucleotide translocator) is an enzyme found in inner membrane (IM) of mitochondria, which imports ADP from cytoplasm through VDAC (voltage dependent anion channel) of outer membrane (OM) of mitochondria and exports ATP generated in electron transfer chain system into cytoplasm (HLA Vieira, et ale, Cell Death and Differentiation, 7, 1146-1154, 2000).
It is also known that ANT playing a key role in energy metabolism of cells is classified into ANT1, ANT2 and ANT3. Particularly ANT2 exhibits low expression rate in normal cells but is highly expressed in cancer cells or similarly highly proliferated cells, which seems to be closely related to glycolysis under anaerobic condition, so that ANT2 is rising up as a new target for cancer treatment (Chevrollier, A, et al., Med. Sci., 21 (2), 156-161, 2005). However, the previous report only suggested the possibility of application to cancer treatment and in fact there has been no reports saying that ANT2 is a target gene which is effective for cancer treatment.
It has been disclosed recently that double stranded RNA (dsRNA) inserted in animal or plant cells could decompose mRNA corresponding to the dsRNA and thereby inhibit a specific protein synthesis, which is called ‘RNA interference’ (Sharp, P. A., et al., Genes Dev., 16, 485-490, 2001). At this time, dsRNA is converted into siRNA (small interfering RNA) by an unknown mechanism and decomposes corresponding mRNA. But, when dsRNA having at least 30 nucleotides is used, non-specific reactions might nullify protein synthesis interruption or at least make the interruption inefficient (Hunter, T. et al., J. Biol. Chem., 250, 409-417, 1975; and Robertson, H. D. and Mathews, M. B., Biochemie., 78, 909-914, 1996). To overcome the above problem, a new technique has been developed to synthesize double stranded siRNA composed of 21 oligomers and to insert the siRNA into mammalian cells to decompose corresponding mRNA to interrupt a specific target protein synthesis (Hutvagner, H. D. et al., Science, 29,834-838, 2001).
In vivo/in vitro experiments have been vigorously performed as follows in order to treat diseases including cancer by synthesizing double stranded siRNA composed of 21 oligomers. For example, [β-catenin that is involved in rapid growth of cancer cells was effectively eliminated from cultured colon cancer cells and mouse colon cancer models by using synthetic β-catenin siRNA (Verma, U. N., et al., Clinical Cancer Res., 9, 1291-1300, 2003; and Annick, H. B., et al., PNAS USA, 99, 14849-14854, 2002).
It was also reported that when multidrug resistance 1 (MDR1) siRNA synthetic oligomer produced to overcome drug resistance of cancer cells, which has been a barrier for chemotherapy, was inserted in MDR1 expressing cells, MDR1 protein synthesis was blocked (Wu, H. et al., Cancer Res., 63, 1515-1519, 2003). When cycline E siRNA synthetic oligomer was treated to cycline E over-expressing liver cancer cells, the proliferation of cultured liver cancer cells and/or liver cancer cells transplanted into a mouse was suppressed (Kaiyi, L. et al., Cancer Res., 63, 3593-3597, 2003).
The above results indicate that siRNA that is overexpressed in cancer cells and at the same time able to interrupt selectively a protein involved in rapid growth of cancer cells can be developed as an effective anticancer agent. Nevertheless, synthetic siRNA allegedly has disadvantages as follows; synthetic siRNA oligomer requires high costs for its synthesis, exhibits low intracellular transmission rate, induces non-specific reaction that might induce cytotoxicity and has short half-life in vivo which suggests that the effect is not constant and thereby the injection has to be repeated. So, in vivo application of synthetic siRNA is limited.
Both viral and non-viral gene carriers that can express siRNA in cells are expected to overcome the said disadvantages of synthetic siRNA oligomer greatly.
The present inventors observed that ANT2 siRNA that was believed to interrupt ANT2 protein synthesis could effectively inhibited the growth of cancer cells where ANT2 was over-expressed, and completed this invention by confirming that ANT2 siRNA can be used as an anticancer agent.